Effects of the histamine H2-receptor blocking drugs burimamide and cimetidine on noradrenergic transmission in the isolated aorta of the rabbit and atria of the guinea-pig

1 In rabbit aortic strips, concentration-response curves to noradrenaline (NA) were shifted to the right in a parallel and concentration-dependent manner by the α-adrenoceptor blocking drug, phentolamine and also by the histamine H₂-receptor blocking drugs, burimamide and cimetidine. Responses to 5-hydroxytryptamine were not affected by these drugs.

2 Burimamide had the properties of a competitive antagonist of noradrenaline, possessing about one-hundredth the potency of phentolamine. Cimetidine was weaker than burimamide and did not fulfil the requirements for competitive antagonism of noradrenaline.

3 In guinea-pig isolated atria, in which noradrenergic transmitter stores were labelled with [³H]-noradrenaline, phentolamine (3 μM), burimamide (30 μM) and cimetidine (30 μM), in decreasing order of effectiveness, each enhanced stimulation-induced efflux of [³H]-noradrenaline, indicating that their blocking effects on prejunctional α-adrenoceptors in this tissue are in the same order of relative potency as on postjunctional α-adrenoceptors in rabbit aortic strips.

4 In the concentrations used (30 μM), neither burimamide nor cimetidine interfered with the neuronal uptake of noradrenaline. Burimamide, and to a much lesser extent, cimetidine, increased the resting efflux of [³H]-noradrenaline from guinea-pig atria.

5 The effect of clonidine, a partial agonist on prejunctional α-adrenoceptors in guinea-pig atria, in increasing stimulation-induced efflux of [³H]-noradrenaline when stimulated with 150 pulses at 5 Hz was blocked by cimetidine (30 μM) and reversed by phentolamine (3 μM) and burimamide (30 μM).

     

Presynaptic α-adrenoceptor blocking properties among tri- and tetra-cyclic antidepressant drugs

1 The effect of various antidepressants (5 × 10^-8 to 2 × 10^-5 M) on the resting overflow of tritium, on the evoked overflow and the contractile response to electrical stimulation (2.5 Hz, 2.0 ms) has been determined in mouse vas deferens previously incubated with [³H]-(—)-noradrenaline.

2 Mianserin and ORG GC 94 produced a concentration-dependent increase of more than two fold in the electrically evoked overflow and the contractile response and, at the highest concentration, slightly increased resting release. These effects were largely unchanged in the presence of a concentration of cocaine effective in blocking noradrenaline uptake (1.1 × 10^-5 M).

3 The ability of phentolamine (1 × 10^-5 M) to increase both the evoked overflow of tritium and the contractile response was greatly reduced when these parameters were already elevated by the presence of mianserin or ORG GC 94.

4 The inhibitory effect of exogenous (—)-noradrenaline on evoked overflow was greatly reduced in the presence of mianserin or ORG GC 94 (4 × 10^-6 M).

5 The inhibitory effect of clonidine on the twitch response of the mouse vas deferens was antagonized by mianserin and ORG GC 94 in a competitive manner (pA₂ values 7.3 and 7.1 respectively).

6 Maprotiline, desipramine and nortriptyline (> 3 × 10^-6 M) produced a parallel fall in both evoked tritium overflow and in the contractile response and increased the resting overflow at higher concentrations. These effects were largely unchanged in the presence of cocaine (1.1 × 10^-5 M).

7 Doxepin, imipramine and iprindole all increased resting overflow at high concentrations (2 × 10^-5 M) but produced only small changes in evoked overflow and in the contractile response at lower concentrations.

8 It is concluded that mianserin and ORG GC 94 produce a blockade of presynaptic α-adrenoceptors which could contribute to an antidepressant effect but that this type of action is not common to all antidepressants.

     

The influence of hormonal and neuronal factors on rat heart adrenoceptors

1 The influence of hormonal and neuronal factors on adrenoceptors mediating increased cardiac force and rate of contraction were studied in rat isolated atria. The pharmacological properties of these receptors were deduced from the relative potencies of agonists and from the effects of selective α- and β-adrenoceptor antagonists. The numbers and affinities of α- and β-adrenoceptors were also determined by radioligand binding to ventricular membrane fragments.

2 Hypophysectomy reduced the inotropic potency of isoprenaline and increased the potency of phenylephrine and methoxamine in left atria. The effect of phenylephrine was inhibited by propranolol less effectively and by phentolamine or phenoxybenzamine more effectively in hypophysectomized than in control rats. The difference in block was smaller at low than at high antagonist concentrations. Similar but smaller changes were observed for chronotropic responses of right atria.

3 The decreased β- and increased α-receptor response after hypophysectomy was similar to that observed earlier in thyroidectomized rats (Kunos, 1977). These changes developed slowly after hypophysectomy (>2 weeks), they were both reversed within 2 days of thyroxine treatment (0.2 mg/kg daily), but were not affected by cortisone treatment (50 mg/kg every 12 h for 4 days).

4 Treatment of hypophysectomized rats for 2 days with thyroxine increased the density of [³H]-dihydroalprenolol ([³H]-DHA) binding sites from 27.5 ± 2.7 to 45.5 ± 5.7 fmol/mg protein and decreased the density of [³H]-WB-4101 binding sites from 38.7 ± 3.1 to 18.7 ± 2.5 fmol/mg protein. The affinity of either type of binding site for agonists or antagonist was not significantly altered by thyroxine treatment and the sum total of α₁- and β-receptors remained the same.

5 Sympathetic denervation of thyroidectomized rats by 6-hydroxydopamine increased the inotropic potency of isoprenaline and noradrenaline and the blocking effect of propranolol, and decreased the potency of phenylephrine and the blocking effect of phenoxybenzamine to or beyond values observed in euthyroid controls. The density of [³H]-DHA binding sites was higher and that of [³H]-WB-4101 binding sites was lower in the denervated than in the innervated hypothyroid myocardium. Depletion of endogenous noradrenaline stores by reserpine did not significantly alter the adrenoceptor response pattern of the hypothyroid preparations and did not influence the density or affinity of [³H]-DHA and [³H]-WB-4101 binding sites.

6 These results indicate that thyrotropin or steroids do not contribute to the reciprocal changes in the sensitivity of cardiac α₁- and β-adrenoceptors in altered thyroid states. These thyroid hormone-dependent changes are probably due to a parallel, reciprocal change in the numbers but not the affinities of α₁- and β-adrenoceptors. Reciprocal regulation of cardiac α₁- and β-adrenoceptors by thyroid hormones requires intact sympathetic innervation but not the presence of normal stores of the neurotransmitter.

     

Mechanisms of inactivation of noradrenaline in the iris sphincter, tracheal muscle and facial artery of cattle: Implications for β-adrenoceptor-mediated responses

1 The role of neuronal and extraneuronal pathways of amine inactivation in regulating the inhibitory actions of noradrenaline was investigated in three bovine smooth muscle preparations in which the primary adrenoceptor is of the β-type.

2 The extraneuronal uptake inhibitor, 17β-oestradiol, sensitized the inhibitory responses to noradrenaline in the facial artery, the iris sphincter and in tracheal muscle preparations, indicating a major role for non-neuronal processes in agonist-inactivation in all three preparations. Cocaine also increased responses to noradrenaline, pointing to a role for neuronal uptake either as a terminating mechanism or as a process limiting access of exogenous agonist molecules to their site of action.

3 Cocaine did not enhance significantly responses to isoprenaline, a potent β-adrenoceptor agonist which is not taken up neuronally. Further, relaxations to metaraminol, a sympathomimetic amine which is taken up extraneuronally, but much less so than noradrenaline, were also less enhanced by 17β-oestradiol in the three preparations tested. These findings support the specificity of action of cocaine and 17β-oestradiol as neuronal and extraneuronal uptake inhibitors in the present experiments.

4 Studies of the uptake of [³H]-noradrenaline revealed that 17β-oestradiol reduced the uptake of amine in the presence of cocaine, confirming a cocaine-resistant site of action for the steroid in all three preparations.

5 It is concluded that extraneuronal uptake sites are located sufficiently close to the β-adrenoceptors to modulate the concentration and duration of action of noradrenaline at these sites of action. It is proposed that in smooth muscles which contain a preponderance of β-receptors, extraneuronal metabolism is a key event in terminating the inhibitory effects produced.

     

Noradrenaline release from slices of the thalamus of normal and morphine-dependent rats

Summary The effect of morphine on potassium-induced stimulation of (³H)-noradrenaline release from slices of the rat thalamus was investigated. The in vitro addition of morphine (10^–6 M) significantly depressed potassium-induced tritium overflow by 42% and this was prevented by the prior addition of naloxone (3×10^–6 M) to the medium. The stimulation-evoked overflow of tritium from slices of the thalamus of morphine-dependent rats was not significantly different from normal controls. Addition of naloxone (10^–5 M) 10 min before exposure of the tissues to 20 mM K⁺ significantly enhanced noradrenaline release from dependent slices. The results suggest that the basic release mechanism may have adapted to the continuous presynaptic inhibition of release by morphine.     

Catecholamine transport in isolated lung parenchyma of pig

1 Lung parenchyma strips of the pig incubated at 37°C with [³H]-(-)-noradrenaline ([³H]-NA) or [³H]-(±)-isoprenaline ([³H]-Iso), accumulated radioactivity via saturable, high affinity uptake processes. Apparent saturation constants (K_m) for [³H]-NA and [³H]-Iso were 1.34 × 10^-6 M and 1.63 × 10^-6 M respectively, while apparent transport maxima (V_max) were 4.86 and 1.63 × 10^-9 mol min^-1 g^-1 respectively.

2 Cellular accumulation of radioactivity from radiolabelled catecholamines was greatly reduced by lowering the temperature to 7°C, pretreatment with ouabain (100 μM), phentolamine (15 μM) or phenoxybenzamine (80 μM). However, accumulation of radioactivity derived from (³H]-NA was inhibited selectively by cocaine (10 μM) and desipramine (1 μM), while normetanephrine (80 μM) and 3-O-methylisoprenaline (50 μM) caused much greater reductions in cellular radioactivity from [³H]-Iso than from (³H]-NA. Taken together with information from kinetic studies, the results indicate that these amines are transported by separate uptake processes.

3 Cocaine (50 μM) which selectively reduced [³H]-NA transport, had no significant effect on the sensitivity (EC₅₀) of isolated parenchyma lung strips of the pig to the contractile effects of cumulative concentrations of NA. The catechol-O-methyl transferase (COMT) inhibitor, U-0521 (60 μM), also failed to alter the potency of NA, while normetanephrine (80 μM) caused a 2 fold decrease in potency.

4 Phentolamine (15 μM), which reduced the cellular accumulation of radioactivity derived from [³H]-Iso by 64%, caused a small potentiation of Iso-induced relaxations of porcine lung strips. Normetanephrine (80 μM) and 3-O-methylisoprenaline (50 μM), which also depressed the accumulation of cellular radioactivity from [³H]-Iso by > 50%, caused rightward shifts in Iso concentration-effect curves as a result of β-adrenoceptor blockade. In sharp contrast, cortisol (80 μM) and U-0521 (60 μM), which caused smaller reductions in the cellular accumulation of radioactivity derived from [³H]-Iso, both caused an approximately 9 fold potentiation of responses to Iso in isolated lung strips.

5 The results indicate that the major sites of uptake and metabolism of NA in porcine parenchyma strip are remote from α-adrenoceptors mediating NA-induced contraction. Similarly, some major sites of uptake of Iso are remote from β-adrenoceptors mediating Iso-induced relaxation. However, β-adrenoceptors are apparently in close proximity to a compartment containing COMT activity.

     

Inhibition by atropine of the increased turnover of noradrenaline in the hypothalamus of rats exposed to cold

1. Small doses of (-)-[³H] noradrenaline were injected into the lateral cerebral ventricles in rats to label radioactively the endogenous noradrenaline (NA) stores.

2. Intraventricular injection of 25 μg atropine methonitrate at the same time inhibited the increased rate of disappearance of [³H] NA from the hypothalamus at an environmental temperature of 9° C, when compared with the values at 24° C, without impairing temperature regulation.

3. At 32° C, 25 μg atropine methonitrate caused a lethal hyperthermia. A dose of 5 μg was not lethal and did not inhibit the increased rate of disappearance of [³H] NA from the hypothalamus.

4. It is concluded that the pathway which stimulates an increased turnover of NA in the cold contains an atropine sensitive synapse but is not the principal pathway of heat production. The increased turnover of NA in the heat probably does not involve an atropine sensitive synapse.

     

Involvement of alpha-receptors in clonidine-induced inhibition of transmitter release from central monoamine neurones

Slices of rat cerebral cortex preincubated with (?)-³H-noradrenaline or ³H-5-hydroxytryptamine were stimulated by an electrical field, and the stimulation-induced overflow of tritium was determined. (1) Clonidine diminished the stimulation-evoked tritium overflow from slices preincubated with ³H-noradrenaline. The degree of this inhibition was greater at a low than at a high frequency of stimulation. (2) A high concentration of clonidine (10^?5 M) did not antagonize the increase of the stimulation-induced overflow caused by 10^?6 M or 10^?5 M cocaine, but abolished the increase caused by 10^?7 M of phentolamine or phenoxybenzamine. In the presence of cocaine, the inhibitory effect of clonidine was reduced. (3) 10^?5 M clonidine diminished the stimulation-evoked overflow of tritium from slices preincubated with ³H-5-hydroxytryptamine. (4) It is concluded that clonidine decreases, and phentolamine and phenoxybenzamine increase, the stimulation-induced release of noradrenaline from cerebral neurones by an activation and a blockade of α-receptors, respectively. A variety of secretory cells (secreting catecholamines, acetylcholine, 5-hydroxytryptamine, insulin or renin) seem to be endowed with structures similar to α-adrenergic receptors, which can modulate the secretion process.     

An examination of the negative feedback function of presynaptic adrenoceptors in a vascular tissue

1 The hypothesis was examined that presynaptic α-adrenoceptors exert a negative feedback function regulating noradrenergic transmission.

2 Renal artery strips from cattle, pre-incubated with [³H]-noradrenaline, were stimulated with 300 pulses at 5 different frequencies, spanning the physiological range, and the efflux of tritium assessed both in the absence and presence of functional presynaptic receptors.

3 Considerable variation in the synaptic level of free and active noradrenaline with increasing frequency was apparent from the rates of development and the magnitudes of the mechanical responses but the overflow of tritium was constant at 1, 2, 10 and 15 Hz and slightly elevated at 5 Hz, providing no evidence for presynaptic modulation of release.

4 Phenoxybenzamine (3.3 × 10^-5 M) enhanced the overflow of tritium most at the lowest frequency tested and to a similar extent at the other test frequencies, except 10 Hz where its effect was slightly reduced.

5 The conditions of the present experiments appeared optimal for the operation of the negative feedback system and the failure to observe an increased effectiveness of the antagonist with increasing frequency indicates that the physiological relevance of such a system is highly questionable and suggests that it may not function at all.

     

Leucine-enkephalin- and neuropeptide Y-modulation of [3H]noradrenaline release in the oviduct of mature and juvenile rabbits

1. The effects of leucine-enkephalin and neuropeptide Y (NPY) on [3H]noradrenaline release induced by electrical field stimulation were studied in the isthmic part of the oviduct of juvenile and mature rabbits. 2. [3H]noradrenaline and total tritium overflow in the presence of cocaine, corticosterone and hyoscine were determined by liquid scintillation spectrometry. 3. Tritium overflow evoked by electrical stimulation (1 or 4 Hz, 1 msec) was calcium dependent. [3H]noradrenaline content (measured by ion exchange chromatography) accounted for 85% of the total tritium overflow. 4. Leucine-enkephalin (1 microM) in the presence of the peptidase inhibitor bacitracin reduced the stimulation-evoked tritium overflow in mature rabbits by 26.1 +/- 1.6% and in juvenile rabbits by 11.9 +/- 1.9%. Naloxone (1 microM) antagonized the effect of leucine-enkephalin. 5. NPY (0.2 microM) reduced the evoked tritium overflow in mature rabbits by 23.4 +/- 2.4% and in juvenile rabbits by 17.2 +/- 4.3%. 6. It is concluded that leucine-enkephalin and NPY inhibited [3H]noradrenaline release in rabbit oviduct and the modulatory effect of leucine-enkephalin depends on maturity while NPY modulation is a more independent system.     

On the mechanism by which veratridine causes a calcium-independent release of γ-aminobutyric acid from brain slices

1 The mechanisms by which veratridine increases the release of γ-aminobutyric acid (GABA) from brain slices have been studied.

2 Exposure of superfused cerebro-cortical, nigral or cerebellar slices to veratridine (5 μM) or KCl (50 mM) caused large increases in the efflux of [³H]-GABA.

3 Reduction of the external Ca concentration [Ca]_o to zero had strikingly different effects on the veratridine and K-evoked release of [³H]-GABA. The K-evoked release from all three areas was greatly reduced in Ca-free medium, but the veratridine-evoked release from cerebeller slices was not affected, and the release of [³H]-GABA from cortical and nigral slices was increased three fold. The potentiation of the veratridine evoked release of GABA which occurred in Ca-free medium was not due to the reduction in divalent ions, because it still occurred in medium in which the Ca was replaced by an equivalent amount of Mg.

4 The veratridine-evoked release of [¹⁴C]-glycine from slices of spinal cord was also significantly increased in Ca-free medium. In contrast, the release of cortical [³H]-noradrenaline and [¹⁴C]-acetylcholine caused by the alkaloid was greatly diminished in Ca-free medium.

5 The veratridine but not the K-evoked release of [³H]-GABA was abolished when the external Na concentration [Na]_o was reduced to zero and by tetrodotoxin (TTX) (0.2 μM). Cl-free medium did not affect the veratridine-evoked release of [³H]-GABA or its potentiation by Ca-free medium.

6 Exposure of the tissue to depolarizing concentrations of external K ([K]_o = 120 mM) did not abolish the veratridine evoked release of [³H]-GABA or its potentiation by Ca-free medium.

7 Pre-incubation of cortical slices with L-2,4, diaminobutyric acid (DABA), or substitution of Na in the superfusion medium with Li, did not affect the veratridine-evoked release of [³H]-GABA, indicating that the alkaloid does not stimulate GABA efflux by a carrier-mediated transport process.

8 Exposure of the tissue to ruthenium red (10 μM) increased the veratridine evoked release of [³H]-GABA in both normal and in Ca-free medium but almost abolished the K-evoked release.

9 It is suggested that veratridine causes GABA release by increasing the permeability of the nerve terminals to Na. In normal medium, the resulting influx of Ca²⁺ ions through voltage-dependent Ca²⁺ channels may be involved in triggering the release of GABA. However, a major part of the GABA efflux appears to be triggered by the release of Ca²⁺ ions from intraterminal mitochondria, which results from the increase in[Na]_i. Since Ca²⁺ ions antagonize the action of veratridine, the potentiation of the drug-evoked release of GABA that occurs in Ca-free medium, might be due to the absence of the antagonistic Ca²⁺ ions. The resulting greater increase in Na entry and [Ca]_i caused by Ca release from intracellular stores, must presumably more than balance the contribution normally made by any influx of extracellular Ca²⁺.

     

Inhibition by apomorphine of the potassium-evoked release of [3H]-γ-aminobutyric acid from the rat substantia nigra in vitro

1 The spontaneous and potassium-evoked release of tritium from the rat substantia nigra prelabelled with [³H]-γ-aminobutyric acid [³H]-GABA were assessed in vitro under conditions of superfusion.     

Opioid receptor sub-types involved in the control of transmitter release in cortex of the brain of the rat

Electrical stimulation (3 Hz, 2msec duration, 5–12 V for 2 min every 20min) of cortical slices from the rat, previously incubated with [³H]noradrenaline, evoked a release of tritium which was inhibited by morphine, normorphine, Tyr-d-Ala-Gly-MePhe-NH(CH₂)₂OH (RX783006) and d-Ala²-d-Leu⁵-enkephalin (pIC₃₀ 5.90, 6.32. 7.45 and 6.74 respectively). Naloxone did not affect the release of tritium when given alone but antagonised the actions of the opioids, giving a K_e value of about 3 nM irrespective of the particular agonist used, which suggests an action at mu receptors. The delta opioid receptor blocker, ICI154129, antagonised the opioids only in large concentrations (K_e 21300nM). In slices previously incubated with [³H]5-hydroxytryptamine, electrical stimulation increased overflow of tritium but neither naloxone nor the opioid agonists affected evoked overflow of tritium at concentrations which were effective in slices incubated with [³H]noradrenaline. It is concluded that stimulation of mu opioid receptors may inhibit release of noradrenaline from central noradrenergic neurones and that these receptors are not present in significant numbers on neurones releasing 5-hydroxytryptamine in the cortex.     

An inhibitory role for noradrenaline in the mouse vas deferens

1 Noradrenaline (0.1-3.0 μM) inhibited the twitch responses to single pulse field stimulation of the isolated vas deferens of the mouse. The higher concentrations of noradrenaline (ca. 0.3-3.0 μM) were required to make the tissue contract.

2 Phentolamine (10 μM) abolished the contractor response to higher concentrations of noradrenaline and antagonized the inhibitory effect of lower concentrations on the twitch response.

3 Propranolol (10 μM) potentiated both the contractor and the inhibitory effect of noradrenaline on the twitch response.

4 Isoprenaline (0.1-3.0 μM) and salbutamol (1.0-3.0 μM) both inhibited the twitch response. Their effects were antagonized by propranolol (10 μM), but not by practolol (10 μM).

5 The effects of uptake₁ and uptake₂ blocking agents were determined. Cocaine (10 μM) reduced the size of the twitch response in 2 out of 4 experiments. Imipramine (0.18 μM) also reduced the size of the twitch, as did oestradiol (3.7 μM) and a combination of cocaine and oestradiol.

6 Contractor responses to exogenous noradrenaline showed tachyphylaxis, but when this was not very marked, the response could be shown to be potentiated by uptake blocking agents.

7 The inhibitory effect of noradrenaline on the twitch response was greatly potentiated by cocaine (10 μM) and much less so by oestradiol (3.7 μM).

8 It is concluded that the transmitter responsible for the twitch response is either an unknown substance released from the sympathetic neurone, or noradrenaline acting upon a receptor with none of the characteristics of known α- or β-adrenoceptors. In either case, noradrenaline can inhibit the output, probably by stimulation of presynaptic α-adrenoceptors.

     

Effects of hypoxia on noradrenaline release and neuronal reuptake in isolated rabbit thoracic aortic strips

Summary To clarify the effects of hypoxia on stimulus-evoked noradrenaline release and on neuronal reuptake of the released noradrenaline, we examined the effects of hypoxia on contraction responses of rabbit thoracic aortic strips to transmural electrical stimulation and on the stimulation-evoked overflow of total [³H] and [³H]noradrenaline from the strips prelabelled with [³H]noradrenaline. This was done in the presence or absence of an inhibitor of neuronal uptake (cocaine). In a medium equilibrated with a gas mixture of 95% O₂/5% CO₂ (control), cocaine doubled the stimulation-evoked overflow of total [³H] and [³H]noradrenaline; there was a concomitant increase (130%) in contractions to electrical stimulation. At 0% O₂ (95% N₂/5% CO₂, hypoxia), cocaine had no significant effects on either the stimulation-evoked overflow of total [³H] and [³H]noradrenaline or contractions. In the absence of the drug, hypoxia decreased the stimulation-evoked overflow of total [³H] and [³H]noradrenaline to 47% and 43%, respectively, of the control values, whereas these values were 31% and 28%, respectively, after exposure to cocaine. The inhibition by hypoxia of contraction responses to electrical stimulation was greater in the presence of cocaine than in its absence. These results show that hypoxia inhibits both noradrenaline release evoked by a given stimulus and neuronal uptake.This work was supported by a Grant-in-Aid for New Drug Development from the Ministry of Health and Welfare of Japan and by a Grant from Smoking Research Foundation, Japan Send offprint requests to S. Miwa at the above address     

Spectrum of the μ-, δ- and κ-binding sites in homogenates of rat brain

1 In homogenates of rat brain, the binding characteristics of tritiated opiates and opioid peptides were examined and the relative capacities of μ-, δ- and κ-binding sites of the opiate receptor determined by saturation analysis.

2 In competition experiments, binding of the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin at the μ-site was displaced by [D-Ala²,D-Leu⁵]enkephalin with rather low affinity (K_I = 12.6 nM) and more readily by the ketazocine-like compounds (-)-ethylketazocine (K_I = 3.1 nM) and (-)-bremazocine (K_I = 0.32 nM), which also displaced the binding of [³H]-[D-Ala²,D-Leu⁵]enkephalin from the δ-site. In contrast, the binding to the κ-site was easily displaced by ethylketazocine (1.0 nM) and bremazocine (0.37 nM) but not by the μ-ligand [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin (K_I = 2000-3000 nM) or the δ-ligand [D-Ala²,D-Leu⁵]enkephalin (K_I > 20,000 nM).

3 The dissociation equilibrium constant (K_D) and the binding capacity (pmol/g) of the μ-binding site were determined with the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin. For the δ-site, [³H]-[D-Ala²,D-Leu⁵]enkephalin was used in the presence of unlabelled [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin in order to suppress cross-reactivity to the μ-binding site. For the estimation of κ-binding, [³H]-(±)-ethylketazocine or [³H]-(-)-bremazocine were used in the presence of unlabelled μ- and δ-ligands for the suppression of cross-reactivities to the μ- and δ-binding sites.

4 In rat brain the capacity of the μ-binding site was 7.3 pmol/g brain, that of the δ-binding site 6.7 pmol/g brain and that of the κ-binding site 2.0 pmol/g brain. Thus, the κ-binding site had the lowest value whereas in the guinea-pig brain the capacity of the μ-binding site was lower than that of the δ- or κ-binding site.

     

The binding of [3h]-oestradiol-17β in the immature rat uterus during the sequential administration of nonsteroidal anti-oestrogens

1 The binding of [³H]-oestradiol-17β (0.08 μg) in the uterus, vagina, liver and heart of immature female rats has been studied in vivo and the effect of daily administrations of the non-steroidal anti-oestrogens, tamoxifen and monohydroxytamoxifen, on the 2 h accumulation of [³H]-oestradiol-17β in the uterus has been determined.     

Rapid, agonist-induced desensitization of alpha 2-autoreceptors modulating transmitter release.

1. The release of previously incorporated [3H]-noradrenaline was investigated in cultures of dissociated chick or rat sympathetic neurones and in cerebrocortical slices from neonatal or adult rats. Noradrenaline, in the presence of 10 mumol l-1 of the uptake inhibitor, cocaine, or the selective alpha 2-adrenoceptor agonist, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 4,304), was applied for different periods of time in order to detect a possible time-dependence of the alpha 2-adrenoceptor-mediated inhibition of electrically evoked tritium outflow. 2. In chick sympathetic neurones, stimulation-evoked overflow was reduced to 30%, 42%, or 56% of control when noradrenaline (1 mumol l-1) was present for 2, 8, or 16 min, respectively. Likewise, UK 14,304 (1 mumol l-1) present for these periods of time reduced 3H overflow to 35%, 51%, and 53% of control, respectively. Addition of 1 nmol l-1 to 10 mumol l-1 UK 14,304 for either 2 or 16 min did not produce significantly different IC50 values, but the inhibitory effects were smaller with 16 min as compared to 2 min exposure at concentrations > or = 10 nmol l-1. 3. In rat sympathetic neurones, noradrenaline (100 nmol l-1) reduced stimulation-evoked overflow to 33%, 56%, or 57% of control, when present for 2, 8, or 16 min, respectively. Addition of UK 14,304 (1 mumol l-1) for these periods of time caused inhibition to 11%, 41%, and 46% of control.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of release and depletion of endogenous noradrenaline on the transmission of impulses in the mouse vas deferens

1 The effects of endogenous noradrenaline released by tyramine and the influence of depletion of the tissue noradrenaline with reserpine and/or α-methyl-p-tyrosine on the twitch responses of the field-stimulated mouse vas deferens have been studied.

2 Tyramine (10-40 μM) inhibited the twitch responses to field stimulation and failed to produce a contraction. The inhibition decreased as the rate of stimulation increased.

3 The inhibition produced by tyramine was antagonized by cocaine (10 μM) and by yohimbine (10 nM), which indicated that it was produced by released noradrenaline acting on presynaptic α-adrenoceptors.

4 Depletion of the tissue noradrenaline by 39% by blockade of the synthesis of noradrenaline with α-methyl-p-tyrosine, was without effect on the twitch response but it reduced the inhibitory effect of tyramine.

5 Depletion of the tissue noradrenaline by 96.5% with reserpine alone and by 99.4%, with a combination of reserpine and α-methyl-p-tyrosine, reduced the twitch responses by approximately 66% and virtually abolished the inhibition produced by tyramine. It also increased the rate of decline of the responses when the tissue was continuously stimulated. The remaining twitch was not antagonized by phenoxybenzamine (15 μM).

6 Residual twitches were bigger in tissues depleted by 99.4% than in those depleted by only 96.5%. This difference was eliminated in the presence of yohimbine (128 nM).

7 It is concluded that inhibition of the twitch responses by tyramine is produced by stimulation of presynaptic α-adrenoceptors and that the twitch response is associated with stimulation of the sympathetic neurone, but that it is not mediated by postsynaptic α-adrenoceptors.

     

The effects of ageing on neuronal uptake of noradrenaline in the rat

Summary We have examined the effects of ageing on the physiological function of the neuronal noradrenaline uptake system by comparing responses to cocaine in young adult (5 month) and aged (22 month) male Sprague-Dawley rats. In rat atria pre-incubated with [³H]-noradrenaline, cocaine (3–30 mol/l) significantly augmented the 2 Hz stimulation-evoked release of noradrenaline in tissues from young but not from old rats. Cocaine (1 mg/kg) produced a greater increase of the pressor response to noradrenaline in young than in old pithed rats. Cocaine significantly increased the tachycardia to noradrenaline only in young pithed rats, but in old pithed rats the duration of the response to noradrenaline was significantly increased. It is concluded that ageing in the rat is associated with a decreased function of the neuronal noradrenaline uptake system, at least in the cardiovascular system. Send offprint requests to J. R. Docherty at the above address